Current studies have on the identification of novel antigens expressed in melanoma and renal cancers as well as the characterization of T cells that recognize epitopes of previously described antigens. In a recent studies, several novel point-mutated genes have been identified as the targets of melanoma-reactive TIL that mediated long terms clinical tumor regression following adoptive transfer. Current investigations are being carried out to develop methods to facilitate the identification of mutated gene products that can be evaluated as targets of tumor-reactive T cells. Recent studies have focused on the use of high throughput sequencing to identify all of the somatic mutations in patient tumors. Minigenes encoding the somatic mutations identified from tumor sequencing have been generated and tested for their ability to be recognized by autologous TIL. Screening of candidate antigens identified resulted in the identification of one or more mutated antigen targets in 12 of the 15 melanoma TIL, as well as in two of the four gastrointestinal TIL that have been screened to date. In recent studies carried out in a patient with cholangiocarcinoma, analysis of reactivity with a mutated antigen was used to guide the selection of TIL used for treatment of a patient who has demonstrated significant regression of multiple metastatic lesions. Ongoing studies are focused on the development of strategies for identifying T cells that recognized mutated antigens in an attempt to develop effective adoptive immunotherapies for the treatment of patients with a variety of malignancies including lung and bladder cancer. Studies carried out in collaboration with the NIH RNAi consortium to develop a novel approach to the identification of human tumor antigens utilizing the transfection of a library of siRNA that target 20,000 human cDNA transcripts resulted in the identification of potential targets that are being evaluated for their ability to be recognized by tumor-reactive T cells. Additional studies have involved the identification of T cell receptors (TCRs) directed against widely shared antigens. Recent studies have focused on the identification of TCRs that recognize cancer/germline (CG) antigens, molecules that are expressed in melanoma as well as a variety of highly prevalent malignancies such as breast and prostate cancer. A TCR that recognizes the NY-ESO-1 CG antigen was used to transduce autologous PBMC, and results of a Phase II clinical trial indicate that approximately 50% of either melanoma or synovial cell sarcoma patients who received PBMC transduced with an anti-NY-ESO-1 TCR demonstrated objective clinical responses. Ongoing clinical trials are being carried out to evaluate TCRs that target both HLA class I and class II epitopes of the MAGEA3 CG antigen. The DNA core facility provides services to many of the members of the Surgery Branch, which includes expertise and reagents used for RT-PCR, quantatitive RT-PCR, gene cloning and RNA/DNA analysis. The FACS Core Facility is currently being utilized for the analysis of T cell populations that are administered to patients as a part of the analysis of ongoing clinical cancer adoptive immunotherapy trials. In addition, the FACS Core is utilized on a daily basis for the analysis of the results of experiments to analyze the results of in vitro experiments to examine factors that influence the phenotype and function of tumor reactive T cells as well as to carry out the separation of cells based upon their expression of a wide variety of cell surface markers. For these studies phenotypic analyses were carried out on a BD FACSCalibur, FACSCantoI, FACSCantoII, as well as a recently acquired LSRFortessa, and cell separations were carried out on a BD FACSAriaII.